Clostridium difficile is the major cause (95%) of disease in patients suffering from antibiotic-associated pseudomembranous colitis and is moderately associated (20%) with patients having antibiotic-associated diarrhea without colitis (Bartlett, J., Clin Gastroenterol. 8:783-801 (1979)). In addition, 19% of patients with chronic inflammatory bowel disease have the C. difficile toxin in their stools and a positive correlation exists between the severity of the illness and the presence of toxin (Trnka, Y., et al., Gastroenterology 80:693-696 (1981)).
There are two toxins, Toxin A and Toxin B, produced by C. difficile (Sullivan, N. M., Infect. Immun. 33:1032-1040 (1982)). Toxin A, or enterotoxin, is responsible for the increase in intestinal permeability associated with disease (Triadafilopoulos, G., et al., Gastroenterology 93:273-279 (1987)). Toxin B, or cytotoxin, is a thousand-fold more potent than Toxin A in triggering the cytotoxic effect in cultured cells (Rothman, S. W., Infect. Immun. 46:324-331 (1984); Sullivan, N. M., Infect. Immun. 33:1032-1040 (1982)).
Toxin A has been reported to have a molecular weight greater than 300,000 kDa while Toxin B is slightly smaller (Lyerly, D. H., et al., Infect. Immun. 54:70-76 (1986)). Further, antisera made against one toxin is not cross-reactive with the other. Thus, the two toxins have distinct biological and serological activity.
Although specific antibiotic therapy exists, a rapid and accurate diagnostic assay for the toxins responsible for the disease does not exist. A rapid latex test has been developed, however, it detects a 43,000 molecular weight C. difficile associated protein (Lyerly, D. M., et al., J. Clin. Microbiol. 26:397-400 (1988)) that is only weakly (67%) associated with clinically defined disease (Peterson, L. R., Am. J. Clin. Path. 87:298-299 (1987)). The in vitro cell cytotoxicity assay is a widely accepted diagnostic for C. difficile associated disease. Unfortunately, this test requires 48 hours to perform and requires technicians skilled in tissue culture. The performance of toxin specific polyclonal based EIA have been disappointing. This is because of the relatively low toxin specific titer and the high level of nonspecific reactivity of the polyclonal antisera (Walter, R. C., et al.. Diagn. Microbiol. Infect. Dis. 5:61-69 (1986)).
Despite their potential utility in the detection of disease, the generation of toxin-specific hybridomas has proven difficult. Lyerly et al., supra. report the production of Toxin A specific monoclonal antibodies. Also, Wilkins et al. in U.S. Pat. No. 4,879,218, issued Nov. 7, 1989, describe the production of Toxin A specific monoclonal antibodies. This patent also describes the production of mono-specific Toxin B antibodies made from polyclonal serum containing both Toxin A and Toxin B antibodies.
A need continues to exist for Toxin B monoclonal antibodies, without cross reactivity to Toxin A of C. difficile.